Welcome to the Tol2kit blog

This is a forum for discussing the Tol2kit; the Lawson lab's related system; and related topics.

The Tol2kit is a set of constructs that can be used with Invitrogen's multisite Gateway recombination-based cloning system to build expression constructs in a Tol2 transposon backbone (derived from constructs from the Kawakami lab).

At present each post serves as a topic under which visitors may post comments, either commenting or asking questions. Please post with your full name and institution (not just your first name or "Anonymous"), as it is helpful for other readers to know who is asking.

To help prevent spam, word verification is turned on: you will have to do a "word recognition" task before you can post a comment.

Thursday, August 30, 2007

Questions on Tol2kit, 8/30/07-11/6/07

Ask general questions about the Tol2kit system by commenting on this post; we will do our best to answer in a timely manner. This posting covers questions from 8/30/07-11/6/07.

30 comments:

Seok-Yong said...

Hi,

I'm wondering if it's OK to use a T3 promoter on pDestTol2pA2 to generate an in situ probe upon completing 3-fragment gateway cloning (p5E-CMV/SP6 + pME-gene of interest + p3E-polyA).

T3 seems to be a bit apart from the stop codon of the gene of interest: two polyA's and Tol2 site in between.

I guess this extra sequence would not affect the in situ efficiency. Am I right?

Just curious! Thanks. SY

Chi-Bin Chien said...

Hi Seok-Yong,

I would advise against using this T3 promoter to make in situ probe. You would be picking up about 900 bp of vector sequence, which adds a lot of potential for background staining. In particular, you would never want to use this probe on any sort of transgenic fish, because it is likely to detect any transgene made with either Tol2 or with Bluescript-based vectors.

Instead, why not just use pCSDest (#201 from Lawson lab: http://lawsonlab.umassmed.edu/GWDestplasmids.htm) to make a pCS2-like plasmid containing your gene of interest? Then you can use T7 to make antisense probe.

Seok-Yong said...

Many thanks for swift response. As you suggested, I will generate another construct using pCSDest. Thanks again. SY

Anonymous said...

Hi,

I am wondering why my LB reaction gave so few clones. The first time it just gave three and the second time there was no clones on the plate. In my first time, I used equal mole DNA (20fmole) for three entry vector and destination vector to set up the reaction which was incubated at RT overnight. But the restriction enzyme digestion showed none of those three clones were correct. So when I repeat the LR reaction, I doubled the vector DNA to 40fmole and, unfortunately,did not see any clones on the plate. Anybody has any idea or suggestion to get this work?

Thanks!

Seok-Yong said...

Hi,

Does anyone have experience with generating DIG-labeled RNA from a gateway vector?

I placed my gene (1.7 kb) on pCSDest and did in vitro transcription (BamHI, T7). The RNA generated was pure, yet the amount was so little. My control plasmid gave me a lot of RNA though. Is it possible that somehow att site could inhibit transcription?

Any input would be appreciated. Thanks. SY

Jenny Jiang said...

Hi Chi-Bin,

Thank you very much for making such useful tools and a wonderful site for people to exchange ideas.

I’m very new to this Tol2kit and multigateway systems. I actually just got the filter paper containing the kits from Will Talbot and tried to grow them up. However, only two out of 7 vectors that I tried had colonies on the plates, another one has only 2 and the rest (4 vectors) didn’t have anything on the plate. What I did was 1) cut the filter paper containing each vector out and put them in 50ul of TE. 2) Took 2ul out of 50 to mix with either Top10 or ccdB E. coli. depend on vector. 3) Spread all 300ul of culture in two plates and leave overnight. I actually tried again with 5ul of DNA for the 4 vectors that I didn’t get anything the first time. Unfortunately, I didn’t have much luck this time either (only two colonies on 1 plate.)

Do you have any suggestions? Is it possible that you could send me some new DNA prep? I read through the Wiki and the blog and couldn’t find any answer to it. The kit I have right now has 2-1-07 on it.

Thank you very much!

Jenny

Chi-Bin Chien said...

Jenny--

Your numbers do seem low, though in the end you only need one correct colony for each clone. Waiting 8 months before recovering DNA from a filter isn't optimal, but probably shouldn't be fatal.

It's not clear from what you wrote how careful you were with microbiological technique. During the transformations, did you follow the heatshock protocol quite carefully, and did you shake transformations in SOC for an hour or two before plating? I assume that you used the right antibiotics (kan or amp/chlor).

It's also not clear which clones grew for you and which didn't. The destination vectors tend to be more difficult to grow, presumably because of the ccdB negative selection cassette (which probably is slightly deleterious even in ccdB-tolerant cells).

I would say that it's worth trying once more, being very careful about how you do the transformations. Potentially you could use electrocompetent cells (higher efficiency).

There is also another copy of the kit at Stanford, in Philippe Mourrain's lab.

If it really proves necessary, we can of course send you another copy.

Jenny Jiang said...

Hi Chi-bin,

Thank you very much for your fast response. The seven clones that I was trying to grow up are:
299 p5E-bactin2 (50ug/ml Kan) (0)
383 pME-EGFP (50ug/ml Kan) (normal)
386 pME-mCherry(50ug/ml Kan) (2)
302 p3E-polyA (50ug/ml Kan) (normal)
394 pDestTol2pA2 (100 ug/ml of Amp, 30 ug/ml of Chlor) (2)
395 pDestTol2CG2 (100 ug/ml of Amp, 30 ug/ml of Chlor) (0)
396 pCS2FA-transposase (100ug/ml of Amp) (0)
I added 5ul (out of 50ul Elute from filter paper) to E. coli and incubated on ice for 30 minutes (ccdB manual said 30 min, so I did it for both Top 10 and ccdB) and heat shock for exactly 30 s. Then cooled on ice for 2 minutes. After adding 250ul of SOC, shake it for 1 hour and plate.

I don’t have access to electrophoration. But I will try again with 10ul of elute to start and 2 hour shaking in SOC before plating. Meanwhile, I will contact Philippe Mourrain to see if they have some to spare.

Thank you very much!

Jenny

Chse said...

Problem with the GAL4-VP16 construct.
I am trying to create a Gal4 line with a promoter I previously tested with GFP. I did the gateway recombination and injected into heterozygous UAS-GFP fish (from ZIRC). The expression in the transgenics is very weak and barely visible. When I compared the sequence of 387-pME GAL4VP16 vector to the one I isolated there was only 1 bp exchange in the attL1 site. I did a BlastX and blastN with the 387 sequence from the website: there seems to be a 40 AA deletion in the 387 Gal4-VP16 compared to the GAL4-VP16 constructs in the NCBI database. Is the vector faulty or this special one just not in the NCBI database? Which vector does the GAl4 VP16 originate from?
Thanks - Christoph

Chi-Bin Chien said...

Christoph--

1) There is in fact a point mutation in attL1 in most (all?) of our middle clones compared to Invitrogen's sequence. We believe this is likely an error in their documentation.

2) We have tested expression constructs made with pME-Gal4VP16 in transients, injecting DNA for both Gal4 and UAS constructs, and gotten good expression.

3) I have heard that the Campos-Ortega UAS:GFP lines (the ones from ZIRC) are rather dim. We have little personal experience though.

4) The Gal4-VP16 cassette is standard--it came from vectors originally published by Reinhard Koester when he was in Scott Fraser's lab. It comparises aas 1-147 of Gal4 (the DNA binding domain), fused to the VP16 activating domain.

So--we are quite confident that the middle clone is okay.

Hope this helps.

Stephanie said...

Hi Chi-Bin and Kristen,

I was just wondering what your procedures are for distributing newer versions of the Tol2Kit to labs that already have older versions (e.g., 1.0)?

Thanks! Stephanie

Chi-Bin Chien said...

Stephanie--

v1.1 is actually a very minor update. If you look at the clone list, the only new clone is pME-MCS. We can send you this one if you need it.

Also, if everyone could please use their full names on the blog we'd appreciate it. It's nice to know to whom we are writing.

Chi-Bin Chien said...

An additional response for Stephanie:

We're in the process of depositing clones with ZIRC; in the future this should be an alternate route for obtaining clones that you want.

We also encourage others to deposit Tol2kit-compatible clones with ZIRC. We are also willing to consider including others' clones in future distributions if you would like.

Stephanie Woo said...

Hi Chi-Bin

For making N-terminal fusions, will v1.2 also come with something like a p3E-MCS? Otherwise, what would you recommend for putting a gene of interest in the 3' position? (so that in the end you'd have promoter: GFP-geneofinterest)

Thanks!
Stephanie

Chi-Bin Chien said...

Stephanie--

To be honest, we build almost all of our entry clones by PCR and BP reactions rather than conventional ligation-based cloning. For fusions, this is especially convenient as it lets you easily control the frame, and minimize the number of additional amino acids.

So to make p3E-your gene of interest, I'd either (1) do a fusion PCR to add a polyA; (2) clone into pCS2+ and then PCR your gene along with the pCS2+ polyA, or (3) omit the polyA altogether and depend on the polyA in the pDestTol2pA2/CG2 backbone.

Tim Cashman said...

Hi,
Great site. I was wondering what the maximum size promoter you have gotten to successfully recombine into pDONR P4-P1R. I have been trying to get a 10kb fragment to recombine, but with mixed results. Do you have any tips? Thanks!

Chi-Bin Chien said...

Hi Tim,

The biggest fragments that I can think of that we have successfully used to make 5' entry clones via BP reaction are about 6 kb. We have done much bigger pieces (up to 14 kb) using conventional subcloning into p5E-MCS or p5E-Fse-Asc. (However, it's hard to get the subsequent multisite LR reaction to work with the 14 kb piece.)

It's hard to make suggestions without more information. What does "mixed success" mean? Have you successfully done smaller fragments?

Possible things to try: (1) use electrocompetent cells to get another order of magnitude in transformation efficiency; (2) add Fse and Asc sites by PCR and then clone conventionally into p5E-Fse-Asc.

Tim Cashman said...

Great, thank you for your help. I have successfully cloned a 5kb piece before. When I've tried the 10kb fragments I do get recombination but the restriction digests are not consistent with the correct product. So I might be seeing some intermediate recombination event. Thank you for your help!

Chi-Bin Chien said...

Hi Tim,

Since you are getting something, another possibility to consider is that big genomic fragments may be prone to internal recombination, since they usually contain repetitive sequence. You might try growing the bacterial plate at 30 degrees or room temperature (lower temperatures will lower the copy number on a pUC19 ori).

Tim Cashman said...

Hello,
That's definitely a possibility and something I'll pursue. Thank you again for your help and running such a great resource!
~Tim

Eve Teh said...

Hi,

Does anyone have the pDONR 221 with attP4r-P3r?

Thanks,
Eve

Chi-Bin Chien said...

Eve-

Not us. Is that something from the new Multisite Gateway Pro 4-insert system?

Eve Teh said...

Yes, but they don't sell the vectors from that kit individually. We were thinking of inserting a P4r-P3r between the IRES and EGFP in the p3E for the purpose of inserting an ORF. We would like to express two different ORFs with the latter one tagged to eGFP. What do you think?

Alyson said...

Hello,

I have been trying to clone a plexin-intracellular gene into pDestTol2pA2 using an lmo2 promoter and the p3E-MTpA. The BP worked great, but the LR has not after a few tries and some alterations. I have used many different competent cell types and I have gotten no colonies to grow. Any advice?

Cheers,
Alyson (UCalgary)

Chi-Bin Chien said...

Eve--

I have two concerns.

First, although I don't know the Gateway Pro system, won't the att sites you are proposing to insert interfere with the attR4-R3 sites in the eventual destination vector (pDestTol2pA2)? You might do better just to add a unique blunt cutter between the IRES and GFP, and add your ORF by conventional subcloning. Do make sure to preserve AUG 11 in the IRES, which is presumably the start site utilized by the IRES.

Second, as you probably know, the IRES isn't as strong as one might like in zebrafish, so you may have problems with getting sufficient expression of the second cistron. Another option would be to use a viral 2A peptide; see this recent paper from the Leach lab.

Chi-Bin Chien said...

Alyson-

What positive controls have you done? Do you know that your prep of pDestTol2pA2 is good? The destination vectors are prone to mutations in the ccdB negative selection cassette. It would be worth doing something like beta-actin/GFP/MT-pA, and also beta-actin/plexin/MT-pA.

Eve Teh said...

Hi Chi-Bin:

Thanks for your most helpful insights. The original idea for putting recombination sites between the IRES and eGFP was so we could switch ORFs easily. We will definitely have to regroup on that and think about the best way to approach this new idea.

Thanks again. Much appreciate it.

Eve

smitcm3 said...

Hi Chi Bin,

I am using the pDONR P4-P1R with a 10kb promoter and also 5kb pieces of each half of the same promoter. I know this vector is likely to self recombine so I have followed the Lawson protocol to select for nonrecombination. I get a very high number of colonies after transforming the BP reaction, but the restriction cuts don't seem to match up with what I want. I have just assumed that it's the pDONR P4-P1R combining with itself. Do you have any tips to prevent this?

Chi-Bin Chien said...

smitcm3--

Two possibilities. One is that you have a bad prep of pDONR P4-P1R; have you had any successful BP reactions with it?

The other is that you're getting internal recombination with your fragments. See comments to Tim Cashman earlier in this thread.

It's probably worth end-sequencing a couple of clones to try to distinguish between these possibilities.

For suggestions, see responses to Tim Cashman.

PS--We'd really prefer it if people would post with their real names.

smitcm3 said...

Thanks for the quick response! I'll send some clones for sequencing and try to figure out whats going on. Sorry about the username it's always been my account names because its easy to remember.

Thanks again,

Chelsey (Emory)